A phosphatidylcholine‐BODIPY 581/591 conjugate allows mapping of oxidative stress in P. falciparum‐infected erythrocytes

Abstract

The chromophore, BODIPY 581/591, has an extended conjugated system that reacts with oxygen centered-radicals leading to changes in its spectral characteristics. Fatty acid-conjugated BODIPY 581/591 transfers readily between membrane bilayers and can be used as a sensor of oxidative stress in cell populations. We report here the use of a phosphatidylcholine (PC) derivative of BODIPY 581/591, which transfers much less rapidly between membranes. This allows the analysis of oxidative stress in individual cells and in different compartments within cells. Quantitative imaging and flow cytometry were used to measure the ratio of fully conjugated to oxidized probe in model systems and in Plasmodium falciparum-infected erythrocytes. We observed an increase in the oxidation of the parasite-associated BODIPY 581/591-PC as the intraerythrocytic parasite matures. By contrast, BODIPY 581/591-PC associated with the erythrocyte membrane experiences a low level of oxidation even in the later stages of parasite development. Treatment with a pro-oxidant compound caused increased oxidation of the probe in the parasite compartment, but less so in the host cell membrane. Conversely, treatment with ferricyanide increases oxidation of the probe in the erythrocyte cell membrane but does not inhibit parasite growth. Chromatographic analysis of the lipids in infected erythrocytes shows no evidence for loss of a-tocopherol or the accumulation of lipid hydroperoxides indicating that, despite the increased oxidative stress, the parasite membranes remain protected from substantial lipid oxidation. We have established BODIPY 581/591-PC as a useful probe of the spatial distribution of oxidative stress in P. falciparum-infected erythrocytes; however, the probe appears to be more sensitive to oxidative damage than endogenous lipids. ' 2009 International Society for Advancement of Cytometry Key terms malaria; Plasmodium falciparum; oxidative stress; BODIPY 581/591-PC; lipid oxidation OXIDATIVE damage to protein and membrane components plays important roles in various diseases and in the aging process (1), and a number of methods have been developed for measuring oxidative stress in cellular systems. In some studies, the levels of oxidized lipids and proteins have been measured directly (2,3). In other studies, secondary reaction products, such as malondialdehyde, have been used as a surrogate measure of lipid oxidation (4). These approaches offer useful insights into oxidative events; however, they usually require physical disruption of the system under investigation.Recently, fluorescence-based probes of oxidative stress have gained popularity as relatively nonperturbing sensors of cellular stresses (5-7). The fatty acid (FA) analogue, cis-paranaric acid (8), has been used as an oxidant-sensitive fluorophore; however, its fluorescence in the UV range is a disadvantage in some applications. BODIPY 581/591 is an alternative probe that can be excited in the visible range and

Atypical lipid composition in the purified relict plastid (apicoplast) of malaria parasites

“…3A), might be required to maintain the multilaminate membrane structure and/or minimize oxidative damage to apicoplast lipids. Blood cell-stage parasites are exposed to high oxydative levels of endogenous reactive oxygen species as a result of hemoglobin digestion in the digestive vacuole and may have evolved a membrane composition that is intrinsically resistant to oxidizative stress (36). The high levels of saturated fatty acids in Plasmodium, and particularly in apicoplast membranes, would complement other well-characterized antioxidant and redox regulatory systems (16,(36)(37)(38).…”

Dual labeling with a far red probe permits analysis of growth and oxidative stress in P. falciparum‐infected erythrocytes

“…Recently, fluorescence‐based probes of oxidative stress have gained popularity as relatively nonperturbing sensors of the cellular environment (14–16). Oxidation‐sensitive lipophillic probes have been used to monitor the level of oxidative stress experienced by P. falciparum ‐infected RBCs (17–19). These studies indicate an increased level of stress in the membrane compartment of mature stage parasites.…”

Inactivation of Malaria Parasites in Blood: PDT vs Inhibition of Hemozoin Formation

“…Besides the decontamination of blood or dialysis, numerous studies have been conducted to understand the physiology of the human malaria parasite Plasmodium, and some PSs have been used. For example, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid (BODIPY FL C 16 ) has been used as a marker for chloroquine resistance [124] or spatial distribution of oxidative stress in infected erythrocytes [125]. Other examples are 5,6-chloromethyl-2,7-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), 5′,6′-carboxy-10-dimethylamino-3-hydroxy-spiro[7H-benzo[c]xanthene-7,1′(3H)-isobenzofuran]-3′-one (SNARF), and 2,7-bis-(2-carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF) for the measurement of the parasite's food vacuolar pH [126].…”